- The incidence of melanoma has been rising rapidly in recent years due to habitual exposure to sunlight. Tanning or pigmentation, resulting from the synthesis and dispersion of melanin within skin, is the main physiological defense against photo-induced injuries, including photocarcinogenesis. An understanding of molecular mechanisms involved in the synthesis of melanin is critical in preventing photo-induced injuries and developing effective therapies. Recent advances in culturing human melanocytes from neonatal and adult skins have allowed for rigorous studies examining molecular mechanisms that regulate human melanogenesis. The applicant's laboratory has identified protein kinase (PKC)-B as a key regulator of human melanogenesis. PKC-B was shown to up-regulate the activity of tyrosinase, the rate-limiting enzyme in melanogenesis. The aim of this proposal, is to characterize the mechanisms through which PKC-B regulates tyrosinase activity. The proposal has the following objectives: (1) Determine if only the B isoform phosphorylates tyrosinase in vitro, then in vivo; (2) determine if phosphorylation leads to an increase in tyrosinase activity; (3) identify phosphorylation site(s) on tyrosinase. In addition, preliminary studies suggest that the cAMP-dependent pathway, previously implicated as the major signaling pathway regulating murine melanogenesis, up-regulates tyrosinase activity by affecting the level of PKC-B. Thus, the final goal of this proposal is to investigate how the cAMP-dependent pathway influences the expression of PKC-B. In order to examine these objectives two cell lines will be used as model systems: cultured human melanocytes from newborn foreskin which are known to express a, b, g, e, z isoforms of PKC, and NP-MM4 human melanoma cells which express all of the above isoforms except PKC-B. To confirm that only PKC-B phosphorylates tyrosinase in vivo, phosphorylation of tyrosinase in NP-MM4 cells permanently transfected with PKC-B cDNA will be compared to that of non-transfected NP-MM4 cells. It is hoped that these studies will reveal a mechanism through which tyrosinase is activated. Furthermore, by examining the influence of the cAMP-dependent pathway on PKC-B, the applicant will begin to understand the role played by the cAMP-dependent pathway in human melanogenesis.